Nucleotide sequence of Klebsiella pneumoniae lac genes.

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1 JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 850-857 Vol. 163, No. 3 0021-9193/85/090850-08$02.00/0 Copyright 1985, American Society for Microbiology Nucleotide Sequence of Klebsiella pneumoniae lac Genes WILSON E. BUVINGER AND MONICA RILEY* Biochemistry Department, State University of New York at Stony Brook, Stony Brook, New York 11794 Received 4 February 1985/Accepted 29 May 1985 The nucleotide sequences of the Kkebsiella pneumoniae lacI and lacZ genes and part of the lacY gene were determined, and these genes were located and priented relative to one another. The K. pneumoniae lac operon is divergent in that the lacI and lacZ genes are oriented head to head, and complementary strands are transcribed. Besides base substitutions, the lacZ genes of K. pneumoniae and Escherchia coli have suffered short distance shifts of reading frame caused by additions or deletions or both during evolutionary divergence from a cominon ancestral gene. Relative to corresponding E. coli sequences, the nucleotide sequences of the lacZ and lacY genes are 61 and 67% conserved, and the lacI genes are 49% conserved. A comp#rison of both nucleotide and amino acid sequences revealed that the K. pneumoniae and E. coli lad genes and lac repressor proteins each are related to the gaLR gene and gal repressor of E. coli to about the same extent. In terms of evolutionary relationships, the divergence of the forerunner of the gaiR gene from an ancestral lac repressor gene preceded separation and differentiation of the K. pneumoniae and E. coli lac repressor genes. The chromosomal lac operon in Klebsiella pneumoniae contained the chromosomal lac genes was cloned, and a (formerly Klebsiella aerogenes) (20) has some characteris- simple restriction map was generated (12). In this study we tics that are similar to those of the lac system in Escherichia determined the complete nucleotide sequence of this HindIII coli, as well as some that are dissimilar (12, 23). As in the E. fragment that contains the' lacI gene, the lac regulatory coli system, isopropylthio-B-D-galactopyranoside acts as an region, the lacZ gene, and part of the lacY gene. The active inducer of the K. pneumoniae operon. Expression of sequences of the structural genes are presented in this paper, the lac operon in K. pneumoniae is subject to the glucose and the regulatory sequence is presented in the accompany- effect, suggesting that, as in E. coli, the complex of cyclic ing paper (6). AMP and its receptor protein acts as a positive regulator. However, unlike E. coli, K. pneumoniae is only weakly MATERIALS AND METHODS lactose positive. Whether the low activity is a consequence of poor expression of lac genes or the production of a less Source of K. pneumoniae lac DNA. A HindIII fragment of active P-galactosidase is not known. K. pneumoniae is also K. pneumoniae lac chromosomal DNA was previously iso- different from E. coli in its response to melibiose, an lated, and its size was estimated by electrophoretic mobility ot-galactoside.' Melibiose induces P-galactosidase synthesis to be 4.95 kb (12). (In this work the true size was shown to in E. coli (1), but in K. pneumoniae it potentiates repression be 4.8 kb.) This fragment was cloned into the pBR322 vector and prevents induction of the lac operon (22). to create plasmid pCR100. Plasmid pCR100 DNA was iso- It seems likely that in the course of evolution of enteric lated, and the K. pneumoniae fragment was excised by bacteria, the lac operons and lacI genes of K. pneumoniae HindIII digestion and then was separated from vector DNA and E. coli descended from a common ancestral source. A by preparative agarose electrophoresis, using standard pro- comparative analysis of the two contemporary lac systems cedures (13). could provide informnation on mechanisms of evolutionary Cloning PstI fragments in phage M13 vectors. The HindIII change in these bacteria, as well as information on the fragment of K. pneumoniae DNA was digested with PstI, molecular basis for the differences in the expression and and the resulting fragments were ligated to the replicative properties of the K. pneumoniae and E. coli lac operons. form of one of the cloning derivatives of phage M13 The genes and proteins of the E. coli lac and gal repressors (Ml3mp8, M13mp9, M13mplO, or M13mpll) that had been are related in both nucleotide and amino acid sequences (25). linearized by digestion with either PstI or PstI plus HindIII. The two repressors share some recognition properties in that After transfection of bacterial strain JM103, individual the respective operators show nucleotide sequence similar- phages were isolated by picking colorless plaques from ity (18) and both repressors appeat to interact with the small plates containing the indicator dye 5-bromo-4-chloro-3- effector molecule methylthio-,-D-galactopyranoside, al- indolyl-o-D-galactoside. The sizes of the insertions were though with different effects since methylthio-P-D-galacto- estimated by gel electrophoresis of PstI or PstI-HindIII pyranosi'de acts as an inducer for the lac operon but as an digests of small-scale replicative form preparations to be 1.7, anti-inducer for the gal operon (5). Sequence comparisons 1.2, 1.0, 0.45, 0.4, and 0.15 kb. M13 derivatives with the could establish the relationships of the K. pneumoniae lac fragment inserted in both orientations were identified by repressor gene to the two E. coli repressor genes, lacI and annealing single-stranded phage DNAs pairwise and deter- gaiR. mining mobility during gel electrophoresis. As preparation for such analyses, previously a 4.8- Sequencing strategy. The nucleotide sequences of the three kilobase (kb) HindIII fragment of K. pneumoniae DNA that smallest fragments were determined without further sub- cloning. Both strands of two of the fragments were se- quenced from both ends by using phage M13 containing the * Corresponding author. insertions in both orientations. The sequence of the smallest 850

2 VOL. 163, 1985 KLEBSIELLA lac GENES 851 fragment was determined in one direction only. The three kb l 2 3 4 largest fragments were sequenced by using the kilobase approach (10). Each fragment was inserted into an appropri- I II z 11 I Y ate M13 derivative in both orientations. A set of deletions was prepared for each, thus yielding two sets of deletions for FIG. 1. Topography of the K. pneumoniae lac operon. The each PstI fragment in which one end of the fragment was locations and directions of transcription of the genes in the 4.8-kb unchanged while truncations were introduced at the other HindIII fragment of K. pneumoniae chromosomal DNA are shown. end. This approach provided clones with overlapping se- The scale (in kilobases) pertains to the DNA strand that encodes the quences for both complementary strands. lacZ and lac Y genes. Preparation of sets of deletion derivatives. The method of Hong (10) was used to generate the sets of deletions. A low concentration of pancreatic DNase in the presence of lacZ and lacY genes are encoded by one strand of the DNA, manganous ion was used to introduce double-stranded cuts and the lacI gene is encoded in the opposite direction by the in random positions in the replicative forms of the M13 complementary strand of the DNA. Consequently, the K. derivatives. The linearized DNA was then cut at a specific pneumoniae lacZ and lacI genes are situated in a head-to- primer-proximal location (14) in the phage DNA by either head relationship. HindIII (for M13mp8 or M13mpl0) or EcoRI (for M13mp9 or The 3,102-bp sequence of the coding region of the lacZ M13mpll) in order to remove fragments of varying sizes gene and the deduced amino acid sequence of ,B- from one end of the insertion DNA. Treated phage DNA galactosidasewere analyzed relative to the corresponding E. then was cyclized with T4 DNA ligase. After transfection of coli sequences. The locations of conserved and differing host bacteria, the resulting phages were propagated from residues were identified in addition to the loci at which one isolated plaques. DNA crudely derived from each phage or the other sequence appeared to contain an excess of culture was hybridized with a replicative form DNA in which residues relative to the other. These features and the com- a full-sized PstI fragment was present but in the opposite plete K. pneumoniae lacZ coding region are shown in Fig. 2. orientation. The hybrid complex was subjected to Si nucle- The C-terminal methionine of the K. pneumoniae amino acid ase digestion to remove single-stranded DNA. The size of sequence aligns with the second methionine (the third resi- the resultant double-stranded insertion DNA was deter- due) of the E. coli amino acid sequence. The nucleotide and mined by agarose or polyacrylamide gel electrophoresis (13). amino acid sequences of K. pneumoniae lacZ that were most From the collection of sized, deleted derivatives, a subset poorly conserved relative to the E. coli sequences were was chosen for sequencing that terminated at intervals of 100 found in the first 45 nucleotides, in a stretch of about 120 to 200 base pairs (bp) throughout the original PstI fragment, nucleotides located about 0.2 of the distance from the and this set was subjected to nucleotide sequencing proce- N-terminal end, in a stretch of about 350 nucleotides at about dures. the 0.7 mark, in a segment of about 100 nucleotides at about Nucleotide sequencing. The dideoxynucleotide chain ter- the 0.8 mark, and finally in a group of 21 poorly conserved mination method was used to determine the sequences (24). nucleotides at the C-terminal end. Several positions of For most experiments, a kit consisting of chemicals, en- unmatched nucleotides representing either additions or de- zymes, and a 15-bp primer was used (supplied by the letions in the K. pneumoniae and E. coli lacZ sequences Bethesda Research Laboratories, Inc.), and the general were observed and are shown schematically in Fig. 3A. A features of the protocol of the manufacturer were followed. cluster of discontinuities was found to lie in a 100-bp region The sequence of each deletion fragment contained se- near the 0.8 mark of the lacZ gene (Fig. 3B). quences that overlapped with the sequences of the adjacent The 48-bp intercistronic region that lies between the stop deletion fragment, thus providing an independent check on codon at the end of the lacZ gene and the methionine codon the sequence in the overlap region. In addition, in almost all at the beginning of the lacY gene was found to contain an cases both strands of the DNA were sequenced, providing inverted repeat sequence of eight bases (Fig. 4). Since no data for the complementary strand. structures characteristic of promoters or operators were Computer analysis. The nucleotide and amino acid se- found in this region, the K. pneumoniae lacZ and lacY quences were managed and analyzed by using the DNA genes, like the lacZ and lac Y genes of E. coli, appeared to be Sequence Analysis Program of Bruce Conrad and David a single operon. Mount (7) and the DNA Protein Sequence Analysis System Only 401 nucleotides at the N-terminal end of the coding of Pustell and Kofatos (21) (International Biotechnologies, region of the K. pneumoniae lac Y gene were sequenced (Fig. Inc.). The E. coli lac operon sequences were obtained as the 5). This was due to the fact that the lacY gene is interrupted file "ecolac" from the DNA sequence data bank at Los by a HindIIl site and the gene lies at one end of the 4.8-kb Alamos National Laboratory. HindIII fragment that was cloned from the K. pneumoniae genome (Fig. 1). By comparison with the corresponding E. RESULTS coli sequences, the partial sequence of the K. pneumoniae The nucleotide sequence was determined for each PstI lacY gene and the deduced amino acid sequence of the fragment derived from the 4.8-kb HindIII K. pneumoniae lac protein were found to be conserved or to differ at the DNA by using the strategy and methodology described residues indicated in Fig. 5. above. The locations and limits of the individual genes of the The nucleotide sequence of the K. pneumoniae lacI gene lac operon within each fragment were detected by searching was aligned and compared with the coding sequence of the for similarities in nucleotide sequences with genes of the E. E. coli lacI gene (Fig. 6). The deduced amino acid sequence coli lac operon. Open reading frames were detected and of the K. pneumoniae lac repressor, comprising 352 amino amino acid sequences were deduced with the aid of com- acid residues, and the amino acid sequences of the E. coli lac puter programs. The geography of the K. pneumoniae and gal repressor proteins were aligned and compared for operon was established in terms of the relative positions of homology (Fig. 7). To optimize the similarities of the repres- the lacZ, lacY, and lacI genes (Fig. 1). In this operon, the sor amino acid sequences, four gaps of one amino acid each

3 852 BUVINGER AND RILEY J. BACTERIOL. A B 1260 1270 1200 1290 1300 1310 2350 2360 2370 2380 2390 AT03 vAJT TCC AAC CEC CAT GAS CAT CAT CAT TTS COC CACTG S STC ACC SCI SAT B T T Ra W ABC ACC USC CSC CAC T Lu 9 If t T R S H T P D F H V A IN R H E H Hl H CD a Vl V T I A D L. Vl 1320 1330 1340 1350 1360 1370 2400 2410 2420 2430 2440 2450 COT EECAA QAC Tw CAC Off CAS ACC ATT ACC CAC CTT AAC CC3C CTO CCA SCO-CCC H T I L LL N R LI P A H PI CA SAC ATT CTO TTS I L L ATS AAM CAB AAC AAC N K A N N TTT MC 6CC STO CUC TSC TCS CAC TAT F N A V R C U H YV 1380 1390 1400 1410 1420 2460 2470 2480 2490 2500 2510 OTT SAT SAS CTT OCC c ceceSAT AAC CTA CCT T TCC CeC CCC A cGM cec TM-TAT GM CTCT AAC TAC Tr CTU TAC UTS OTC ST V F D E L a D N L fPj s R A M R IN Yl E IL Cl N IR Y S L Y V V Dl 1430 1440 450 1460 1470 1480 2520 2530 2540 2550 2560 C8C TCAA CTO UAC Gss AMT USC AST TCT CTT ACS CCC UCA 9cc CUT TTe CCS TC8 SAA 9CC AAT ATT SM ACC CAC ese ATS eTC CCS ATO AAT COO CTS TCC WAC CAT C R 0 19 [ D 8eU S S L T A R L P 9 E A N I E T H U M V P M N R L US D D P 1490 1500 1510 1520 1530 1540 2570 2560 2590 2600 2610 2620 *~~~~~~ ATO CGA STO eTO ACG CAS SAT CTA CCS Tsc CDC USC ACS CCT Uce 1e CTA CCA GM TTC STA_SM A C6GTCCC OCC ATC AUC AACC3C AAC n R V V T D LPL DljC R T P V P N A IN L P AI F [] A IR jVT R j IV N Nl 1550 1560 1570 1560 1590 1600 260 2640 2650 2660 2670 268 CAT eTC ATT ATC ATC TOO TCGS CTO UBC AAC SAG TCC USC UC USC BSC AAC CAC 8 * * TM CAB ATO SAO Uc TAT SAC UCe CCe ATC TAC ACC AAC OTC CBC TAT CCC ATC SAC H E Y8IV A P I Y T N VI R Y P Il D C I I I S L N E el e a 1610 1620 1630 1640 1650 2690 2700 2710 2720 2730 2740 ACC ACC CCA CCe COs OTO CCe SAs SAT AAC CCS ACC esC TSC TAC TCC CTO CAC TTT am Ae CTO TAC CAC TOO CTO AMA CeC AAC SAT CCe ABC CUT CCe UTe CAS TAC SAG T T [P R E D I P T e C Y S H El E IA L YI H [H JL E3 R N ID P S R P V a Y EI 1660 1670 1680 1690 1700 1710 2750 2760 2770 2780 2790 AC:TOTT OAe CAA AAC fTC BAT C AB ATT ATT TTC BBC USC USc UeC UcU BAT ACC ACC UCC ACC SAT ATT ATC TUT CcG ATS TAC 6cC CGC STC E D E] AC T ED] R E N Is Q I a I I F D a Vl a15 aB A D T T A T D I I C P " Y A R V 1720 1730 1740 1750 1760 1770 2600 2610 2820 2830 2840 2860 AAC TCI WCA TTT CAT CTO TUG TC AAT UC STU sTC BC TA TCU CAS SAC AsT ez CGC SAC CAB CCS ATC CCe ece STA CCC AMTUGeGU ATC AAM AMA TUG ATC ABC IN 8 F H L N C N GI V w Vv y sIla D SO E R ID Q PlI IF A VlIP rR -VWl e I K K H|l1In 1780 1790 1800 1910 1820 2860 2870 28Go 2890 2900 2910 Cc: CTe c eT cA CAU z AUG CTS ATC CTT T3C GAS TAC eCC eCS ATO eeC AAC ce CTO IRN L PI A cesce TTC A IF SAT D CTC L ASC S CCC P TTC F CTU L CUT R CC P USC a SAC D AAC IN CeC CTO LLP e E I ^ JR P L I L C E Y A CAC H A H e. Ni 830 is"o lsow 160 [email protected]@ 1070 2920 2930 2940 2950 2960 TOC eTe ATe OTC ATO CDC TUS ASC eCC CTO SAM TUG ATU TUG ABC CTC GMc AAC TTC SAC CAs sAT 6CC SAT TAC TUG CAS 9CC TTT CIcEAS TAT CCG cee CTO CAB C IV " lJ " IR " 81 A 1 1 S| IL E D D " [B L Tl N I AlD IY i Q A F Rl E IY P R L Q 1690 1900 1910 1920 1930 1940 2970 2980 2990 3000 3010 3020 AI TIC CC 8eec TTm ATC TOO eCC SAC CAS eCS ATC C8C AAA ACC TTT eCC SAC BUC ft AEC I F[ TCU EWAh TUG CILCST N IK P 0 C MA COU R CTA L U U F I N D M A D 0 A I RJE3 T F A D [ 1950 1960 1970 1980 1990 3030 3040 3050 3060 3070 3060 TEC GAC eTe CAU TTe ACG CCA eCC CTT SAC eCC q TAT CsC SAC GeC ACT CTO CAB AGC OTC eec TMS ec TAT USc Usc SAC TTT GUT SAT AA8 CCT AAC SAT CUC CAS TTC C m , T P A L D A Y R D T L 6 v U F7 A Y U U D F U D K P N D Rf FI 2000 2010 2020 2030 2040 2050 3090 3100 3110 3120 3130 eU Csc ACC ATC McO ACT McO Wcs CTT eCC OM CTC AMC TC asm sTT TOT ATO AAC MT CTG OTO TTT ccC T cec AC8 a TCU C S V A T I SM E A T SM E A A L A M El [eE C M N e L V Fl P ?D0 R T P H P |L V E AI 2060 2070 2060 2090 2100 2110 3140 3150 3160 3170 3160 3190 5 * * * Am CcT Tgg cec eec ame eAe CAS TTC sCC Uge COU CAB CM TTA UST ACC CCe ecc U TAT m ACGCTO ICTOTEcR J TCU CCe CTG COG STU E El 6CC K H1A IY F 0 F T 2 L Y T 6 P L V L Ml R PE F A R Q L El T P R 2120 2130 2140 2150 2160 2170 3200 3210 3220 3230 3240 3250 5 * * * * 5 I,R_C CeC ATC ATC [AC A CT CCA ACC SAT BM OTC eTU CeC TUG CAS ACE T STE SAT V S -AG CeC a R USC CAC e H TAC L e CS AA CE 1 "= SAT TTC TCC .Ue V D F A eGM AA ft I I E Y L F RI P T iID AAC N I V V R J[ G 2160 2190 2200 2210 2220 3260 3270 3280 3290 3300 3310 * * * * * * m CAB MO GCC 8AA =_ SM TAT CAC S0 [C AC ACC CTO UC CTO CCC AC CCe z ec CAC TUG AC 6c ACC c TT TAT 8am T ACC IL A A J E Y H D T L a L P jPj T A N P E T CIY R A V V1 T O L 3320 3330 3340 3350 3360 2240 2250 2260 2270 2280 * * * S 2230 * *5 SATG * ATC T M I 16 F Rl R IC GM wAUC SAC MOU ?IL A TC CT A CCT CC cEI TM COC 10 R eB .D E AA re CTO L ASA E JQ E A TM ATC E 8,J D E T Q L D 8 I L IP lj3 WC R 2290 2300 2310 2320 2330 2340 * * 8 SA OTT GCC SAT CTSCI3 CUT CTC gAC C8T AM CCe CTO CTS ATC C8C USC OTT E JfJ A D L R L IN K P L L I R Vq FIG. 2. Nucleotide sequence and deduced amino acid sequence of the K. pneumoniae lacZ gene. The numbers coincide with the kilobase coordinates in Fig. 1 and locate the nucleotides of the lacZ gene relative to the total sequence of the 4.8-kb HindIll fragment. The K. pneumoniae lacZ amino acid sequence was aligned with the E. coli sequence. The C-terminal methionine of the K. pneumoniae sequence corresponds to the second methionine in the E. coli sequence (the third residue from the C-terminal end). The K. pneumoniae amino acid residues that were conserved with respect to the amino acids of the E. coli sequence are shown in boxes. The excess nucleotides in the K. pneumoniae nucleotide sequence that have no counterpart in the E. coli sequence are also shown in boxes. The nucleotides in the E. coli sequence that have no counterpart in the K. pneumoniae sequence are shown as circled numbers that indicate the number of excess bases at each indicated position. The E. coli lacZ nucleotide sequence was determined by Kalnins et al. (11). were introduced. Three of these occurred at the same summarized in Table 1. Pairwise comparisons of amino acid locations in both of the lacI repressor sequences, and the sequences, as well as nucleic acid sequences, revealed other was in the galR sequence. similar degrees of conservation of the E. coli and K. pneu- The degree of conservation of the nucleotide sequences moniae lac repressors and lacI genes. When the 5' and 3' and the amino acid sequences of the three repressors are overhangs were omitted from consideration, 49% of the

4 VOL. 163, 1985 KLEBSIELLA lac GENES 853 c 3380 of these genes, the lacZ and lacY genes are moderately 3370 3390 3400 3410 3420 conserved, and the trpA gene is the most highly conserved. A CTB BAB OTB ACC CAB CCCSSS SSS GCG6BT CAS e Approximately equal evolutionary distances were de- A V W L L E Mf T [IQ A T A W S E Al 3430 3440 3450 3460 3470 3480 tected between the E. coli lacI and K. pneumoniae lacI GAB CAC CGC GTC 6CC TOB CAA CAB TTT CCC CTB CCC 8CC CCB CTC BBC TBC Cos CBC sequences in relation to the gal repressor sequences. In both E CM R V JA Q F P (3 P A P Be C R R cases, 23% of the amino acids were conserved, and 30 to 3490 3500 3510 3520 3530 31% of the nucleotides were conserved (Table 1), but the CA CCB TBT CTB CCe BCB CTC CCB GAT CTT ATC BTC P P C L P rA L [M D [D I V BAT GAB BTC TB CAB AT D E V N B conserved amino acids were not identical in the two cases. 3540 3550 3560 3570 3580 3590 Even so, a subset of 24 amino acids has been conserved in all C8C ecc TCB CAA Tec ACC ATC BAT CBM ACe CTB CITe CsC TB three repressor molecules. R A s a C TL I D8 R T L R N The patterns of codon usage were determined by using a 3600 3610 3620 0 3640 0 computer program for the lacI and lacZ genes of K. pneu- TCB OTT S V jJE GT8 B CAB GAB CAB CT TTBO ACT CCC CTO COT BOC CAB I ATT Q E 1Q L L T P L R D Q F I A moniae and E. coli. A comparison revealed that the fre- 3660 3670 3680 3690 3700 quency of use of codons in the two lac genes is similar for E. CCB CTC BAC AAC BAC ATC 888 BTC ABC BAA BTA BAB CT ATC A CCC AAC CTC coli and K. pneumoniae, except that utilization of synony- P L D N D I -6 V S E1 V E JR I D P N A WI mous codons with guanine or cytosine in the third position is 3710 3720 3730 3740 3750 3760 greater in the K. pneumoniae genes (73% for lacZ, 78% for BTB SAS CeC T7O ABA ABC 8CC BBC CTB TAC BAT CTT OAM ecB CAC T8C BTC CAB TIC lacl) than it is in the E. coli genes (57% for lacZ, 59% for r-V E R l R B L [ D L [E_ H C V lacl). 3770 3780 3790 3800 3810 BAT D CAB CeC CTB BCA AAT BAA ACC Q R NL A E T C1BTC V M3C D TUC CDC TUB CAC TAC CTO CDC C R N H Y L R DISCUSSION Heterogeneity in conservation of the nucleotides and (i) BWC BAA 38A* ATE :HjjV (DT (a) ATCTTC T 10 T ACT e WC aACC ) amino acids encoded within the lac genes of K. pneumoniae e E V I V s H N R H nH F T A D T and E. coli undoubtedly reflects both stringent and loose 3890 3900 3910 3920 3930 relationships between primary structure and function. Fu- CTO COS TTe BcU L R L A BTlq [ aDB BC e CBs R A BAA ACC CTO CcS CCB CTB CCO E T L P L [ P V L C TC sion proteins with partial deletions of E. coli 0-galactosidase 3940 3950 3960 3970 3980 390 have shown that the first 26 amino acids are dispensable for CAC TTC seeL IH OTO GBAT CAB CAB Bce CCe BTB ABC T8 CTB GBT CTU MI8 catalytic function of the enzyme (3, 17). Consistent with this isB F E V 0D Q Q A P [E] s r L e L B1 observation, the first 16 amino acids of the K. pneumoniae 4000 4010 4020 4030 4040 4050 enzyme are poorly conserved with respect to the E. coli CAT H BAG E AAC TAC CCC BAC C IN Y P D RI CUB ABC ABC R B B IA C Fl Mcc CUC TU OA CAB A r -I E B sequence. It has been shown that the first eight amino acid 4060 4070 4080 4090 4100 residues of the lac permease of E. coli are dispensable (2). AM CP3.~IMBCB OM TB ACC eCCCP C ATC T!C AC8 BAA AAC UBC CTO CUC TOT Consistent with this finding, the initial 14 N-terminal amino I.~~~.jiAA 7IT P YII T~ T N acid residues of the galactoside permease encoded by lacY 4110 4120 4130 4140 4150 4160 genes of K. pneumoniae and E. coli are poorly conserved. BAT D CABeCe CB O BAC TUB O A [3 D a eCUC TUB CAC ATC ABC Rft H I B CAT H F1 H CAC TTC 7CC B Conservation is observed in the remaining sequence. The 4170 4180 4190 4200 4220 4210 clusters of phenylalanine residues and the high proportion of 8TT CAB CCA TUB ABC ACC CST TO OM ACC SAC CM TM CAC AAB ATI CAB other hydrophobic amino acid residues that characterize the V a P N B T R B L n E Tl D H N H K M O E. coli lac permease protein (4) similarly characterize the 4230 * 4240 8 4250 * 4260 4270 * S deduced amino acid sequence of the K. pneumoniae lac 8CC BAA BAC A5J D [ 878T TUB ATC ACC CTC ]V I T L B CTU CAT ATU88 L [H M B| OTe V 18 BBC GAT e DI permease (Fig. 5). The 48-bp nucleotide sequence that lies 4280 4290 4300 4310 4320 430 between the stop codon of the K. pneumoniae lacZ gene and SAC TCC T Te CTB B ACC9CC ABC CAB TUB CTC CT CA ACS TUB CAB the initiation codon of the lac Y gene and contains an inverted D 5 T IP S_ I L a N L IL 5 T N a repeat sequence which is high in G + C content (Fig. 4) is 4340 S 4350 * 4360 * 4370 * 4380 * 4390 * unlike the corresponding E. coli sequence, which contains T A6TC TCA TTB COT ABC S L R S CTT TAB TCC 6T7 88 L - CCB ACA 8CC CCC ACC CCA CAB an A + T-rich palindrome (4). Although this intercistronic JE V 4400 4410 sequence is not a good candidate for a rho-independent ACA BAA TAB CAA O8 ATC transcription termination sequence, since it lacks the char- acteristic run of thymine residues on the distal side of the hairpin loop, nevertheless the palindromic G + C-rich struc- ture seems likely to play a role in regulating the rate of expression of the lacY gene relative to the lacZ gene. nucleotides of the K. pneumoniae and E. coli lacI genes The nucleotide sequences of the coding portions of the K. were conserved in relation to each other, and 40% of the pneumoniae and E. coli lacI genes are related, with an amino acid residues were conserved. The greatest concen- overall level of conservation of the bases of 49% (Fig. 6). tration of conserved amino acids was found between amino The deduced amino acid sequence of the K. pneumoniae acid residues 7 and 90, where 46 of 83 amino acids (55%) are repressor is similarly related to the E. coli lac repressor and the same, whereas the least conserved region was found is 40% conserved (Fig. 7). The regions that are most highly between residues 91 and 149, where only 12 of 59 (20%) are conserved among the K. pneumoniae lac, E. coli lac, and gal the same. The extents of conservation of nucleotide se- repressor proteins are the first 60 amino acids, constituting quences and amino acid sequences in the lacZ, lacY, and the headpiece that in the E. coli lac repressor is known to lacI genes are presented together with comparable data for bind to DNA (3), and the residues between amino acids 180 the trpA gene (19) in Table 2. The relative frequencies of and 300, in the body of the protein (Fig. 7). These commonly utilization of identical, synonymous, and replacement held residues presumably participate in establishing the codons were determined for each of the four gene pairs. general features of the three repressor proteins, the ability to These data showed that the lacI gene is the least conserved bind to DNA, and the ability to interact with galactose or

5 854 BUVINGER AND RILEY J. BACTERIOL. A +14 +3 N +23 +3 +3 Y +9 +3 +1+7 1+1 KP C-rr r -, I . Il z lI I +>2z+t+1 .~~~~ +2 +1 +2 +3 +1 +1+2+4+ +15 B 3794 l 3895 % GTAGT .CGGCGCATGCAS KP CTCGTCACTGCCGCTGGCACTACCTGCGCGGCGAAkGA TTG~TCACT TGGC ACCCTGCGGTTGGCvGTG EC 11 I 11 11111 I 111111 111111111 11 11 11111 11 CTGATTACGACCGCTCACGCTGGCAGCATGGGGAAAA-AJAT 7T G GTTAGGT~,AAATGGCGATTACCGTT C A CAA CT T c 3789v 3890 FIG. 3. Additions or deletions or both in the K. pneumoniae lacZ gene relative to the E. coli lacZ gene. (A) Schematic locations of unmatched nucleotides in the two lacZ genes. At each position indicated, the numbers indicate the number of nucleotides for which there is no counterpart in the other sequence. This is formally equivalent to gaps of the designated size in the opposite gene. (B) Mismatches for the aligned sequences of both the K. pneumoniae and E. coli lacZ genes in the region that contains the cluster of addition-deletion sites that are located near the 3' ends of the genes. KP, K. pneumoniae; EC, E. coli. small galactoside effector molecules. Presumably the unique is four amino acids longer than the E. coli gal repressor and specificities toward operator sequences and small effector two residues longer than the E. coli lac repressor (Fig. 7). At molecules of the three repressor proteins are determined by the C-terminal end, the gal repressor is 16 amino acids some of the amino acids that differ among the three se- shorter than the E. coli lac repressor (26), and the K. quences. Detailed studies have been done on the effects of pneumoniae lac repressor appears to be eight residues amino acid substitutions resulting from alterations within the shorter than the E. coli lac repressor and eight residues E. coli lacI gene (15, 16). Alterations at amino acid residues longer than the gal repressor. However, the location of the C 92 through 97 (i'C mutants) were found to alter the interaction terminus of the K. pneumoniae repressor, as deduced from of the E. coli lac repressor with isopropylthio-p-D-galacto- nucleotide sequence data, may be an artifact of cloning. The pyranoside such that isopropylthio-p-D-galactopyranoside nucleotide sequence of the lacI gene is located at one end of becomes an anti-inducer rather than an inducer (16). Of the the cloned 4.8-kb HindIlI fragment. The cloned lacI gene corresponding six amino acids in the K. pneumoniae repres- was oriented so that the 3' end of the gene abutted the sor sequence, only one is conserved, and none is conserved pBR322 vector stop codon that follows the HindIII site, in in the E. coli galR sequence. The dissimilarities in the frame with the coding sequence of the lacI gene. Since primary sequences in this region could be related to the HindIII cut the K. pneumoniae DNA on the 5' side of the known differences in the physiological responses of the three genomic lacI gene stop codon, the position of the genomic operons to the small effector molecules. Reverse phenotypes stop has not yet been established. If in fact the K. pneumo- are known to exist in that melibiose is an inducer for the lac niae lacI gene is artificially truncated in the cloned HindIII operon in E. coli but is an anti-inducer with repression- fragment, nevertheless the cloned sequence is adequate for potentiating action in K. pneumoniae, just as methylthio-P- function since expression of the associated lacZ gene in the D-galactopyranoside is an inducer for lac in E. coli but is an plasmid is regulated, requiring action of the inducer anti-inducer for the gal operon. In vitro mutagenesis exper- isopropylthio-,3-D-galactopyranoside to relieve repression iments would address the proposition that amino acids 92 (12). through 97 are critical in the specificity of interactions of A point-to-point comparison of the amino acid sequences repressors with galactosides. of the three repressor genes showed that the two lacI genes Other differences among the amino acid sequences of the are entirely congruent, but that shifts have occurred relative lac and gal repressors concern the lengths of the proteins to the sequence of the galR gene (26). The four gaps that and the existence of internal additions or deletions. At the relate the two lacI repressors to the galR repressor (Fig. 7) amino termini of the proteins, the K. pneumoniae repressor indicate that during evolutionary divergence of the gal and AC G A C-G G-C G-C G-C G-C G-C T-A Z-**TAATCC CCCACAAACAGAATAACAAGGGATCATG... Y 4361 4411 FIG. 4. Nucleotide sequence of the intercistronic DNA between the lacZ and lacY genes. The stop codon for the lacZ gene is shown at one end, and the ATG methionine codon of the lacY gene is shown at the other end. An inverted repeat sequence is shown in the form of a base-paired hairpin loop. The numerical coordinates coincide with those of Fig. 1.

6 VOL. 163, 1985 KLEBSIELLA lac GENES 855 3760 3770 3790 3790 4420 4430 440 4450 4440 4470 * 3900 K.p. ATG CCO COT COT ACC OCA ACC CTO GA0 GAT OTC GCO COC COC G6 COT OTC CCA GCA E. c. GTG CCA GTA ACG TTA TAC GATBTC BCA GaB TAT 0CCGGT OTC TCT TAT AT6 IT6 ATC AM TTC TCA6 CT6 6C CCA CA C66 CAT AT TTC 6TC TAT TTC CTI 3810 AAA 3820 3830 3840 3650 3860 N V K F EL A P E NI GAC GGT CTC COC COO BTG AAC COT CCT GAB OTO GTC TCA CTG CST GCC ACC CBC BAG CAB ACC OTT TCC COC GTOGTO AAC CAB 6CC AGC CAC GTT TCT BCO AAA ACO COG BAA 4410 449 4500 4510 4520 4530 3970 3990 3690 3900 3910 3920 CIS TTC TIT TIC ITT TAC CAT TtC ATT AT CCI TIT TTT C6 IT6 166 TCS 6CC TAC TIC CAB STG ATC COCBCG ATG CAG McO CTO CAC TAT OTO CCT AAC CBC TCB BCB CAB CT6 GAA GCG 6CO ATG BCG BAG CTO AAT TAC ATT CCC AAC C6C STG SCA CAA HIF A Y f P F AAA GT6 F F F tY CAA IL NIFI I 3930 3940 3950 3960 3970 450 4550 4540 4570 4560 45" CTTecc 9GC AAA BCMO C6 CCC TCC ATC CTS ATT ACC 9CC TCC STO ACB CTG CAC CTO BCG GOC AAA CAB TCO TTB CTO ATT BOC BTT GCC ACC TCC AST CTG GCC CT6 CAC 3990 3990 4000 4010 4020 4030 CIO IC$ 6SC BIt MC CAT CtA ACT AM AC 6SM ACC O1 ATC ITtTIC CI TCT ATC TNO s * $ A V[JD EEL T t E IT 6 1J v 0S S BCC CCC TCO CAB ATC 9CC GCG CCG TCS CAA ATT OTC 9CC BCB ecO ATA AAB A9C CAT SCO ATT AAA TCT CeCBCC 9CC ABC CTB3 CAT CA CT AA BAT CAA CTO GOT BCC AOC 4040 4050 4060 4070 4060 4090 4600 4410 4420 4430 4440 46 STOGCB ATA GCG ATB CCG OcM CABBCC BAT TTT STC GCB CTB CAB 9CC CBB CTB BAC GTB STO BTG TCG ATO STA CBA ABC GC BTC BCC TGT AAA BCB GCG GTB CAC 1TA TKCcA ATIT T C CTI ATl TCS OAT M6 CTC BC CH CKC C 6TIC A I I FI Pi v 6 LI N 1S I K L J L RI 4100 4110 4120 4130 4140 GAG CTO CeC 6CC CAB CAT ATT 966 ATC STt ABT CT9 CCG CT6 BAG ABC 9CC CBC ,BT AAT CTT CTC GCG CAA CBC OTC ABT G6 CTG ATC ATT AAC TAT CCB CTB 6AT SAC CAB 4460 4470 446 469 470 4710 4150 4160 4170 4190 4190 4200 AM CAT CtO Ct TN ACC ATTAC ITA CtI TtA ATt CtI tC 6 CCA TTC ttt T t ACT OCC GAB COG CTB BTO CAA IAC AAT CCB BAT ATO TBC CTB TTT CTC eCC GaT BTC TO "AA GCT 9CC TBC ACT AAT OTT CCG BCS TTA TTT CTT GAT OTC IL L U1 t TV L E] I LIF A P F I I fl BAT GCC ATT OCT 4210 4220 4230 4240 $ 4250 4260 s $ 4720 4730 4740 470 4740 4770 TCC CCG TCT GAC CAB ACA BAG 9CC BAT CCC OTC TGC ATC AAC TBC STO CBC TTC AST ATT ATT TTC TCC CAT BAA SAC CAC CBC SAC 9AC GOT TBC ACB CBA CTG 9CC 4270 4280 4290 4300 4310 4320 65T TIC tCC CC6 CII CI CA6 AT MI ATT ATC KCT MT tC6 CT6 66 ATC TA 1 sTe CDC CAC CTO TOO GAB ATe CAT COC BAA TTT GOT CTO CTO OCB GSA CCB v I I p L N I A L 6Y TBC GBC BTB BAG CAT CTS OTC GCA TTs GOT CAC CAB CAA ATC GCO CTG TTA BCB GGC CCA 4330 4340 4350 4360 4370 4760 47" 4U00 4610 BAA ABT TCO GTT TCC 9CC COT CTS COT CTC BCC ABC TOO CUC BAB 9C9 CAC TTO ABT TTA ABT TCT GTC TCG BCG COT CTB COT CTB GCT sBC TeO CAT AAA TAT CTC ACT COC COuWAlT OTT TTC TeAC ST CCI WC SIC A'K TI I V El IT 6 .[a I 4390 CTO AAT ATi AAT CAA ATT CAB CCO 9CC 4390 CeC 4400 TCT ACT ACS STO TTT BBC OAK TOO ABC 9CC ATA BCG BAA COG OAA 99C 9AC TOO ABT 4410 4420 eCC GCC ATB 4430 ABC BBC TOG TCC GOT TTT FIG. 5. Nucleotide and deduced amino acid sequence sequence 4440 4450 4460 4470 4460 4490 of the N-terminal end of the K. pneumoniae lacY gene. The CAB AAA ACT TTC GAB CTC CTC CAC CTB CAB CCS COO ATC ASC ATA OTO OTO eCC GCA numerical coordinates coincide with those of Fig. 1. The amino CAA CAA ACC AT9 CAA AT9 CTO AAT GAB BBC ATC OTT CCC ACT 9CS ATO CTO ecc BTT acids that are conserved relative to the residues of the E. coli 4500 4510 4520 4530 4540 galactoside permease are shown in boxes. The locations and quan- AAC BAT CAB ATO CBs CTC IOOC 6Ts CTC ABC WcS CTB GCC CAB CTC AAT CBC ABC BSC tities of excess nucleotides in the E. coli sequence that have no AAC GAT CAs ATO 9CG CTO GGC SCA ATG CsC 9CC ATT ACC GAB TCC BGG CTG CBC OTT counterpart in the K. pneumoniae sequence are shown as circled 4550 4560 4570 4590 4590 4600 numbers. The nucleotide sequence of the E. coli lacY gene was AGC CAB GCB BTA TCB ST9 ACC GBC TAC BAT SAC ACC 9CC SAC ABC CTT TAC TTC CAB determined by Buchel et al. (4). GBT GCG BAT ATC TCG STA STO G6A TAC SAC GAT ACC BAA SAC ABC TCA TST TAT ATC 4610 4620 4630 4640 4650 4660 lac repressor genes, four amino acid codons either were CCG CCB CTC ACC ACS BTB BCG CAB GAC TTC SAT CTO TTS USC AAA AGO BC6 6TG GA CCO CCO TCA ACC ACC ATC AAA CAB BAT TTT CGC CT6 CTO MBO CAA ACC ABC GT8 GAC added or were deleted from either the precursor to the lacI 4670 4680 4690 4700 4710 genes or the precursor to the galR gene. COO CTG ATT GCC CTG ATO SCB 6CC CCG CAB CTB COO ATC CBC GAG CTO CTO CCG ACC The evolutionary distance between the gaIR gene and CGC TTG CTG CAA CTC TCT CAG 88C CAB aCB 9T9 AA9 (IC AAT CAB CTO TTO CCC 6TC either of the lacI genes is greater than the distance between 4720 4730 4740 4750 4760 4770 the two lacI genes, both in terms of sequence homologies COO TCA CTC CTG ATC GTG 9TG CGC AAA ABA CAB AAA TC6 ACC BCC ACC TOO CTO CCT BTC SC6 CCC 6CC A,AT 9CC ACB GC8 GAG CAA ACC GAC COO 9CC TCT CAB CCC CAB CBC and in terms of the gaps (Table 1). These relationships 4780 4790 4800 4810 indicate that an ancestral repressor gene gave rise to a lac GCG CAG CTS GCG BT8 type of repressor gene and a gal type of repressor gene at a ACC CTG AAA CTG AAG CTT GCG TTG GCC GAT TCA TTA ATB CAG CTG MA CrA CAB OTT TCC CBA CT9 GAA ASC B CA time that preceded the divergence of the K. pneumoniae and FIG. 6. Nucleotide sequences of the K. pneumoniae and E. coli E. coli lac repressor genes (Fig. 8). Therefore, the separation lacd genes. The nucleotide numbers shown are those that were and differentiation of the enterobacterial lac and gal repres- assigned to one of the strands of the 4,812-bp HindIII fragment of K. sor genes appear to have occurred in an ancestral pneumoniae chromosomal DNA that was cloned in plasmid pCR100 enterobacterium, preceding the subdivision and emergence (12). Numbering is clockwise relative to the clockwise numbering of of the two enteric genera Escherichia and Klebsiella. vector pBR322. The K. pneumoniae lacI gene resides at the clock- wise end of the HindIII fragment. The sequence of the E. coli lacI gene was determined by Farabaugh (8). K.p., K. pneumonlae; E.c., E. coli. TABLE 1. Pairwise comparisons of lac and gal repressor genes IILevel of similarity (%) Codon usage in the K. pneumoniae and E. coli lacZ and Gene pair A No. of lacI genes and in the N-terminal parts of the lac Y genes were acids Nucleotides gaps determined by a computer sequence analysis (data not shown). Comparisons of the codon usage data revealed that E. coli lacI-K. pneumoniae lacI 40 49 0 codons in the K. pneumoniae genes all exhibit a higher G + C E. coli lacI-E.coli galR 23 31 4 content in the third position than is found in the correspond- K. pneumoniae lacI-E. coli galR 23 30 4 ing codons of the E. coli genes. The same relationship was

7 856 BUVINGER AND RILEY J. BACTERIOL. 10 20 30 40 50 60 E. c. lcI VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLL K.p. /c I MPRRTATLEDVARRGRVPADGLRRVLNRPEVVSARTREQVIRAMIALHYVPNRSAQLLAeKAAPS E. c. 90/ R NATIKDVARLAGVSVATVSRVINNSPKASEASRLAVHSAMESLSYHPNANARALA4QTTLT 70 80 90 100 110 120 130 140 * J * * * * * * IGVATSSLA1HAPSQIVAAIKSRADQLBASVVVShVERSGVEACKAAVHNLLAQRVS6LIINYPLDDQDAIAVEA I6LITASVTLHAPSQIAAAIKSHASLHQLEVAIAtPAQADFVALOARLDELRAQHIRGVIVSLPLESATAERLVQ VGLVV6DVSDPFFAMW.AVEQVAYHTWNFLLI6NGYHNEWKERQAIEQLIRHRCAALVVHAKMIPDADLASLMK 150 160 170 180 190 200 210 * * * S * * * ACTNVPALFLDVSDQTPINSIIFSHED6TRL6VEHLVALGHQQIALLA6PLSSVSARLRLAGWHKYLTRNQIGPI DNPDMACLFLDVSPEADVCCVRFDHRDGC8ACVRHLWEM6HREFGLLAGPESSVSARLRLASWREALHSLNIARS QIPGNVLINRILPGFENRCIALDD RY6AWLATRHLIQQGHTRIGYLCSNHSISDAEDRLQ63YYDALAES6IAAN _ _ ~ ~ .i _ _ _ _ _- _ _ _ _ _ 220 230 240 250 260 270 280 S S S * * * * A EREGDWSAMS6FQQTTM 0LNEGIVPTAfLVANDQ MALGAMRAITES3LRVGADISVVGYDDTEDSSCYIPP T TVFGDWCAASGWQKTF ELLHLQPRISAIVVANDQ MALGVLSALAQLNRSGSQAVSVTGYDDTADSLYFQPP DRLVTFGLPDES8GEQAlATELLGR8RNFTAVACYNDASMAAGAMGVLNDNGIDVPGEISEIGFDDVLVSRYVRPR 290 300 310 320 330 340 350 STTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMOLARQVSRLESGQ LTTVAQDFDLLGKRAVERLIALMAAPQLRIRELLPTRLIVRQSAWPVAAAEDRQQTLAQLKALVEKL LTTVRYPIVTMATQAAELALALADNRPLPEITNVFSPTLVRRHSVSTPSLEASHHATSD FIG. 7. Amino acid sequences encoded by the K. pneumoniae and E. coli lacI genes and the E. coli galR gene. See references 8 and 26. The numbers are the numbers deduced for the K. pneumoniae lac repressor amino acid sequence. Conserved amino acids relative to the K. pneumoniae sequence are underlined. Gaps were introduced at positions 165, 216, 233, and 251 to optimize sequence matching. K.p., K. pneumoniae; E.c., E. coli. found for the trpA genes of K. pneumoniae and E. coli (19). be characteristic of the level at which the genes are ex- These data are consistent with the greater G + C content pressed (9). In K. pneumoniae, the lac genes utilize codons (56%) of K. pneumoniae genomic DNA compared with the in the manner of moderately expressed genes, like the trpA lesser content (51%) of E. coli DNA (25). gene of K. pneumoniae (19) and unlike the lpp gene that The frequency of use of certain codons has been shown to exhibits codon distributions that are characteristic of a high level of expression (27). Point mutations are not the only kinds of changes that alter the coding regions of genes during evolutionary divergence. TABLE 2. Conservation of nucleotide and amino acid sequences Additions or deletions can introduce gaps in the alignment of of K. pneumoniae and E. coli genes two related sequences that in molecular terms might reflect % of con- % of % of re- No. of errors in replication or errors in sister strand recombination. % of con- % of The lacZ gene of E. coli (Fig. 2 and 3) embodies more Gene served nu- served i dentical synony- place- additions cleotides amino ac- codons mous ment or dele- interruptions than are observed in the K. pneumoniae and E. ids codons codons tions coli lacY, lacI, and trpA genes (Table 2). Clusters of the lacZ 65 61 34.5 26 39.5 21 addition-deletion events within the K. pneumoniae lacZ lacYa 67 65 31.2 33.8 35 2 gene or E. coli lacZ gene or both probably identify portions lacI 49 40 18 22 60 0 of the ,B-galactosidase polypeptide chain whose primary trpAb 76 87 41 46 13 0 sequence is not critical for subunit interaction or catalytic a Data for 400 nucleotides at the N-terminal end of the coding region of the activity. From an evolutionary standpoint, a single addition la Y gene. or deletion event can be substantially more powerful than a b Data from reference 19. single base substitution event. As is the case for many loci in

8 VOL. 163, 1985 KLEBSIELLA lac GENES 857 7. Conrad, B., and D. Mount. 1982. Microcomputer programs for DNA sequence analysis. Nucleic Acids Res. 10:31-37. 8. Farabaugh, P. J. 1978. Sequence of the lacI gene. Nature (London) 274:765-769. 9. Grantham, R., C. Gautier, M. Gouy, M. Jacobzone, and R. Mercier. 1981. Codon catalog usage is a genome strategy modulated for gene expressivity. Nucleic Acids Res. 9:r43-r74. 10. Hong, G. F. 1982. A systematic DNA sequencing strategy. J. Mol. Biol. 158:539-549. 11. Kalnins, A., K. Otto, U. Ruther, and B. Muller-Hill. 1983. Sequence of the lacZ gene of E. coli. EMBO J. 2:593-597. anc. 12. MacDonald, C., and M. Riley. 1983. Cloning chromosomal lac genes of K. pneumoniae. Gene 24:341-345. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular 106(0) cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Messing, J., and J. Viera. 1982. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction frag- ments. Gene 19:269-276. I'(.p. lGCI E.c. focI E.c. ga/R 15. Miller, J. H., C. Coulondre, M. Hofer, U. Schmeissner, H. FIG. 8. Evolutionary branching order of the K. pneumoniae and Sommer, and A. Schmitz. 1979. Genetic studies of the lac E. coli lacI genes and the E. coli galR gene. anc, ancestral; E.c., E. repressor. IX. Generation of altered proteins by the suppression coli; K.p., K. pneumoniae. The numbers of calculated nucleotide of nonsense mutations. J. Mol. Biol. 131:191-222. replacements are shown for each branch, and the numbers in 16. Miller, J. H., and U. Schmeissner. 1980. Genetic studies of the parentheses indicate the numbers of gaps introduced. lac repressor. X. Analysis of missense mutations in the lacI gene. J. Mol. Biol. 131:223-248. 17. Muller-Hill, B., and J. Kania. 1974. Lac repressor can be fused the K. pneumoniae and E. coli lacZ genes, when additions to P-galactosidase. Nature (London) 249:561-563. and deletions introduce gaps that are not multiples of three 18. Musso, R. R. Di Lauro, M. Rosenberg, and B. deCrombrugghe. into the colinear alignment of the genes, then the reading 1977. Nucleotide sequence of the operator-promoter region of frame is changed so that the amino acid sequence down- the galactose operon of Escherichia coli. Proc. Natl. Acad. Sci. stream of the gap is altered. The new frameshift persists until U.S.A. 74:106-110. similar events restore the initial reading frame as a conse- 19. Nichols, B. P., M. Blumenberg, and C. Yanofsky. 1981. Com- quence of achieving an algebraic sum of a multiple of three. parison of the nucleotide sequence of trpA and sequences immediately beyond the trp operon of K. pneumoniae, Salmo- Thus, a potential for major change exists as a consequence nella typhimurium and E. coli. Nucleic Acids Res. 7:1743-1755. of a single molecular event. 20. 0rskov, I. 1974. Genus VI. Klebsiella Trevisan 1885, p. 321-324. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's ACKNOWLEDGMENTS manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. This work was supported by Public Health Service grant GM28926 21. Pustell, J., and F. C. Kafatos. 1984. A convenient and adaptable from the National Institutes of Health. package of computer programs for DNA and protein sequence management, analysis and homology determination. Nucleic Acids Res. 12:643-655. LITERATURE CITED 22. Reeve, E. C. R., and J. A. Braithwaite. 1973. The lactose system 1. Barkeley, M. D., and S. Bourgeois. 1978. Repressor recognition in Klebsiella aerogenes V9A. lII. Specific repression of the lac of operator and effectors, p. 177-220. In J. H. Miller and W. S. operon by melibiose and raffinose. Genet. Res. 22:217-221. Reznikoff (ed.), The operon. Cold Spring Harbor Laboratory, 23. Reeve, C. R., and J. A. Braithwaite. 1974. The lactose system in Cold Spring Harbor, N.Y. K. pneumoniae V9A. IV. A comparison of the lac operons of 2. Bocklage, H., and B. Muller-Hill. 1983. lacZ-- Y fusions in Klebsiella and E. coli. Genet. Res. 24:323-331. Escherichia coli. Eur. J. Biochem. 137:561-565. 24. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- 3. Brake, A. J., A. V. Flower, I. Zabin, J. Kania, and B. Muller- ing with chain terminating inhibitors. Proc. Nati. Acad. Sci. Hill. 1978. 3-Galactosidase chimeras: primary structure of a lac U.S.A. 74:5463-5467. repressor-p-galactosidase protein. Proc. Natl. Acad. Sci. 25. Shapiro, H. A. 1970. Distribution of purines and pyrimidines in U.S.A. 75:4824-4827. nucleic acids, p. H83-H85. In H. A. Sober (ed.), Handbook of 4. Buchel, D. E., B. Gronenborn, and B. Muller-Hill. 1980. Se- biochemistry: selected data for molecular biology, 2nd ed. CRC quence of the lactose permease gene. Nature (London) Press, West Palm Beach, Fla. 283:541-545. 26. von Wilcken-Bergmann, B., and B. Muller-Hill. 1982. Sequence 5. Buttin, G. 1963. Mechanismes regulateurs dans la biosynthese of galR gene indicates a common evolutionary origin of lac and des enzymes du mdtabolisme du galactose chez Escherichia coli gal repressor in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. K12. J. Mol. Biol. 7:164-182. 79:2427-2431. 6. Buvinger, W. E., and M. Riley. 1985. Regulatory region of the 27. Yamagata, H., K. Nakamura, and M. Inouye. 1981. Comparison divergent Klebsiella pneumoniae lac operon. J. Bacteriol. of the lipoprotein gene among the Enterobacteriaceae. J. Biol. 163:858-862. Chem. 256:2194-2198.

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